The invention relates to regulatory sequences directing tissue-specific expression of a heterologous gene in a plant, and more particularly to DNA promoter sequence capable of conferring germination-specific expression of a gene in plant tissue.
An important goal of plant biotechnology is to genetically engineer plants so they have a new or improved trait or characteristic. Initially, transformation was developed in model dicot plants. Monocot plants, which include all the major cereal crops, were more difficult, and the first successful transformations, in rice and maize, were not reported until the late 1980s. Although consistent transformation of the more recalcitrant cereals such as wheat and barley have only been achieved very recently, it has been shown that a homozygous transgenic barley line can transmit a heterologous gene over three generations to all progeny plants (Jensen et al., 1998, Hereditas 129:215-225).
While tissue-specific, heterologous gene expression in plants can be achieved, the current stage of genetic engineering methodology does not offer the means of targeting where introduced DNA sequences are integrated into the chromosome; integration into plant chromosomes appears to be more or less random.
This invention relates to transgenic plants and involves a method of generating transgenic plants with controllable gene expression. Particularly, the invention relates to transgenic plants that have been modified such that expression of a heterologous introduced gene can be limited to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location, or a combination of these situations. More particularly, attention has been given to produce transgenic cereal plants of the grass family; common cereal plants include barley (Hordeum), wheat (Triticum), rice (Oryza), maize (Zea), rye (Secale) and sorghum (Sorghum).
A desired trait or characteristic is introduced into the plant by incorporating into the plant""s genome a gene that encodes the polypeptide that confers the desired trait or characteristic. DNA sequences that regulate the expression of the gene must also be introduced into the plant in conjunction with the desired gene. For heterologous expression, the regulatory sequencexe2x80x94such as a sequence often called a gene promoter, or simply a promoterxe2x80x94directs transcription of a large number of RNA molecules from the operably linked heterologous DNA sequence, which serves as a template. Each plant gene comprises a promoter sequence to which specialized proteins bind and activate the gene. For example, specific nucleotide sequences within the promoter are recognized by RNA polymerase molecules that start RNA synthesis. After primary transcription, a second class of signals leads to the termination of RNA synthesis and the detachment of RNA polymerase molecules from their respective DNA templates. The RNA chains, which may undergo further processing, e.g. removal of intron sequences and attachment of poly(A) tails, can in turn serve as templates for the synthesis of specific polypeptide chains.
The selection of a promoter is often a critical factor in obtaining expression of a heterologous gene. A promoter can function as a constitutive promoter or as an inducible promoter. Constitutive promoters are those which are capable of expressing operably linked DNA sequences in all tissues of a plant throughout development. Even though providing constitutive expression of a gene in plants is often desirable, it is also desirable in some instances to direct expression of a gene to particular tissues and/or time of development in a plant. Tissue specific promoters are capable of selectively expressing heterologous DNA sequences in certain plant tissues. Tissue specific promoters may also be inducible, e.g. activated by application of external or internal inducing agent, such as gibberellic acid and abscisic acid which are known to exercise important control at the transcriptional level over a-amylase gene expression in aleurone cells (Skriver et al., 1991, Proc. Natl. Acad. Sci. USA 88:7266-7270; Gubler and Jacobsen, 1992, Plant Cell4:1435-1441).
Of particular interest to the present invention are tissue specific promoters. These promoters can be fused with a heterologous DNA sequence and used to transform a plant cell to create a transgenic plant that selectively expresses the heterologous DNA in a specific tissue. Several promoters are currently being used for tissue-specific, heterologous gene expression in monocot cells. For example, the promoter regions from genes coding for hydrolases have been used to direct germination-specific expression of a heterologous DNA sequence in transgenic monocot cells (see Skriver et al., 1991, supra); Wolf, 1992, Mol. Gen. Genet. 234:33-42; Mikkonen et al., 1996, Plant Mol. Biol. 31:239-254; Jensen et al., 1996, Proc. Natl. Acad. Sci. USA 93:3487-3491; Jensen et al., 1998, supra; U.S. Pat. No. 5,712,112).
A variety of plant promoters with different characteristics and which are effective in different plant species and/or organs is desirable in order to bring potential applications of transgenic plants into practice. Since the task of successfully cloning and demonstrating the utility of a highly expressed promoter is formidable, the use of systems for transient expression of heterologous genes in protoplasts has proven ideal to predict whether a gene construct will function during plant germination (Jensen et al., 1996, supra).
Since the location of the heterologous gene in the host""s DNA can affect the efficiency with which it is expressed, it is often necessary to produce many individual transgenic plants to ensure that an effective line with the desired characteristics can be selected from them. These plants are then bred conventionally.
In view of the widespread occurrence of xcex1-glucosidases in higher plants and their potential importance in carbohydrate metabolism, it is surprising that there are only a few reports on xcex1-glucosidase genes. These include cDNA sequences for xcex1-glucosidases of barley (Tibbot and Skadsen, 1996, Plant Mol. Biol. 30:229-241), spinach (Sugimoto et al., 1997, Plant Mol. Biol. 33:765-768), and potato (Taylor et al., 1998, Plant J. 13:419-425), as well as a genomic sequence of an xcex1-glucosidase gene from Arabidopsis thaliana (Monroe et al., 1997, Plant Physiol. 115:863; GenBank Accession No. AF014806).
Only a few genomic DNA sequences encoding plant cystatins have been described, including the genomic sequence for rice cystatin (Kondo et al., 1989, Gene 81:259-265), the sequence for potato cystatin (Waldron et al., 1993, Plant Mol. Biol. 23:801-812), and the sequence for maize cystatin (Abe et al., 1996, Biosci. Biotech. Biochem. 60:1173-1175).
In no previously reported case has an xcex1-glucosidase gene promoter or a cystatin gene promoter been used to direct heterologous expression in plants. One object of the present invention is therefore to provide an xcex1-glucosidase gene promoter or a cystatin gene promoter from an industrially important organism, and utilize the promoter to direct expression of heterologous protein in monocotyledonous grass plants, including the cereals.
Novel germination-specific promoters have now been identified and isolated from the barley genome. Two such useful regulatory sequences described and claimed herein are the promoters of the xcex1-glucosidase gene and the cystatin-1 genes from barley, which are useful to express a desired heterologous gene at high levels in the aleurone tissue of germinating kernels, particularly barley. In a preferred embodiment, these promoters are used to induce expression of heterologous genes in the aleurone tissue of kernels during germination, including the process of malting, for example, in the production of a brewed product such as beer.
An isolated nucleotide sequence comprising at least 1930 base pairs (hereafter abbreviated bp) upstream of the translational start site of the sequence encoding the xcex1-glucosidase gene was found to be a useful tissue specific promoter (FIG. 2), [SEQ ID NO:1]. A useful promoter fragment of 984 bp in length was isolated (FIG. 2), [SEQ ID NO:2] and used to direct tissue-specific expression of a heterologous gene in monocotyledonous plant cells and plant tissues.
An isolated cDNA sequence encoding barley cystatin-1 was cloned and characterized (FIG. 7) [SEQ ID NO: 5]. Moreover, the gene promoter and protein coding region was cloned (FIG. 8), [SEQ ID NO: 15]. This sequence includes 1508 bp upstream of the translational start site, which was found to be a useful tissue specific gene promoter [SEQ ID NO: 14]. The accumulation of cystatin-1 mRNA transcripts in barley kernel during germination is particularly enhanced under malting conditions.
The present invention provides nucleic acid sequences defining the xcex1-gluocosidase gene promoter region [SEQ ID NOS: 1 and 2], and the cystatin-1 gene promoter region [SEQ ID NO: 14].
The invention also includes a chimeric gene comprised of the xcex1-glucosidase gene promoter or the cystatin gene promoter operably linked to a DNA sequence comprising an open reading frame that is heterologous to the gene promoter. Preferably, the protein coding region of the gene is also operably linked to a 3xe2x80x2 non-translated polyadenylation region. The invention further includes transformed plant cells and plant tissues comprising this chimeric gene; and transgenic plants comprising a gene sequence which expression is regulated by an xcex1-glucosidase gene promoter or a cystatin gene promoter, where said gene sequence is heterologous to the gene promoter. In a further embodiment, the present invention is directed to a recombinant vector, preferably a plasmid, comprising the recombinant DNA molecules described above.
Such recombinant DNA molecules, or vectors containing the DNA molecules, are introduced into plant cells so that the gene promoter preferentially directs expression of the heterologous gene in aleurone cells. Having disclosed the barley xcex1-glucosidase gene promoter and cystatin-1 gene promoter sequences and their ability to direct germination-specific expression of heterologous genes in a plant, those skilled in the art can readily appreciate the identity of other equivalent nucleotide sequences (i.e. gene promoters) capable of directing expression of similar xcex1-glucosidases and cystatins. Thus, the scope of the subject invention includes not only the specific nucleotide sequences disclosed herein, but also structurally and functionally equivalent nucleotide sequences directing expression of molecules with the same xcex1-glucosidase or cystatin activity. These equivalent molecules can be identified, for example, by cross-hybridization to parts of the barley xcex1-glucosidase gene promoter sequence [SEQ ID NO:1] as shown in FIG. 1B, or the cystatin-1 promoter sequence [SEQ ID NO: 14] under conditions of stringency as is well understood in the art and described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
The invention further comprises methods for generating a transgenic plant by introducing a chimeric gene, preferably together with a selectable marker gene, into plant cells, plant tissue and plants where the chimeric gene is comprised of an xcex1-glucosidase gene promoter or a cystatin-1 gene promoter, a gene sequence which is heterologous with respect to the promoter, and preferably a 3xe2x80x2 non-translated polyadenylation region. Plant cells are cultured in a growth medium preferably containing a selection agent to identify those plant cells with the chimeric gene. The transformed plant cells are then regenerated into whole plants.
The present invention provides transformed plant cells, plant tissues, and transgenic plants expressing heterologous proteins as directed by an xcex1-glucosidase gene promoter or a cystatin-1 gene promoter.
The invention further includes transformed plant cells and tissues, and transgenic plants expressing heterologous proteins that are targeted for secretion by use of an xcex1-glucosidase signal sequence, e.g., barley xcex1-glucosidase signal sequence [SEQ ID NO:4], or by use of a cystatin signal sequence, e.g. barley cystatin-1 signal sequence [SEQ ID NO:8]. Thus, the present invention can be used to secrete heterologous proteins from kernel cells and tissues, e.g. barley kernel cells and tissues. Specifically, the functioning of the heterologous constructs as described in the Examples below proves that the signal peptide of barley xcex1-glucosidase can be used to confer secretion of heterologous proteins from barley cells to the extracellular space.
Controlling the expression of genes that direct expression of heterologous proteins has particular utility in industrial applications, such as malting of cereals. Accordingly, the present invention is particularly useful in the brewing industry.